Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes. These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins. By forming a continuous protein pathway across the membrane, these proteins enable the specific molecules to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer of the plasma membrane.
There are two major classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins bind the specific molecule to be transported and undergo a series of conformational changes in order to transfer the bound molecule across the membrane. Channel proteins, on the other hand, need not bind the molecule. Instead, they form hydrophilic pores that extend across the lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Transport through channel proteins occurs at a much faster rate than transport mediated by carrier proteins.
Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the membrane are referred to as voltage-sensitive ion channels.
Ion channels serve numerous physiological functions in excitable and nonexcitable cells (Catterall, W. A., Science 242: 50-61 (1988); Lester, H. A., Annu. Rev. Physiol. 53: 477-496 (1991); Jan. L. Y. et al., Annu. Rev. Physiol. 54: 537-555 (1992)). They transmit electrical signals to generate physiological cell responses. With electrophysiological recording techniques, a variety of ionic currents in many kinds of cells have been observed (Catterall, W. A., Science 242: 50-61 (1988); Lester, H. A., Annu. Rev. Physiol. 53: 477-496 (1991); Jan. L. Y. et al., Annu. Rev. Physiol. 54: 537-555 (1992)). The importance of these ionic currents has been demonstrated by pharmacological approaches using either naturally existing ion channel toxins or inorganic and organic ion channel blockers (such as local anesthetics). The essential physiological roles of ion channels in normal cellular functions have been further strengthened by the link of diseases to defects in ion channel genes (Catterall, W. A., Science 242: 50-61 (1988); Lester, H. A., Annu. Rev. Physiol. 53: 477-496 (1991); Jan. L. Y. et al., Annu. Rev. Physiol. 54: 537-555 (1992)).
Over the past few years, molecular biological studies have revealed a large number of ion channel genes that could be responsible for the observed ionic currents (Pongs, O., Physiol. Rev. 72:S69-88 (1992); Perney, T. M. et al., Semin. Neurosci. 5:135-145 (1993); Chandy, K. G. et al., In CRC Handbook of Receptors and Channels ed. North, R. A. (Boca Raton, Fla.: CRC), pp. 1-71 (1995); Deal, K. K. et al., Physiol. Rev. 76: 49-67 (1996)). For example, there are more than 20 genes that have been cloned coding for voltage-gated potassium channels (Pongs, O., Physiol. Rev. 72:S69-88 (1992); Perney, T. M. et al., Semin. Neurosci. 5: 135-145 (1993); Chandy, K. G. et al., In CRC Handbook of Receptors and Channels ed. North, R. A. (Boca Raton, Fla.: CRC), pp. 1-71 (1995); Deal, K. K. et al., Physiol. Rev. 76:49-67 (1996)). Just within the Kv1 subfamily of the voltage-gated K+ channels, there are at least seven members, and most of them (except Kv1.4) generate similar delayed-rectifier K+ currents. Moreover, different potassium channel subunits can co-assemble to form heteromultimeric channels (Isacoff, E. Y. et al., Nature 345:530-534 (1990); Ruppersberg, J. P. et al., Nature 345: 535:537 (1990); Christie, M. J. et al., Neuron 2:405-411 (1990)). Finally, the native complex of voltage-gated K+ channels is also composed of accessory β-units and these β-subunits could convert the delayed-rectifier currents into rapidly inactivating A-type K+ currents (Rettig, J. et al., Nature 369:289-294 (1994)).
Antibodies have previously been used in functional studies of channels. Antipeptide antibodies, made against regions between S5 and S6 transmembrane segments of domains I and IV of the sodium channel α-scorpion toxin to sodium channels reconstituted in phospholipid vesicles or synaptosomes (Thomsen et al., Proc. Natl. Acad. Sci. USA 86:10161-10165 (1989)). It was not shown whether these antibodies could block sodium currents. An antipeptide antibody, by binding to a region in the intracellular loop between domains III and IV, slows sodium channel inactivation (Vassilev et al., Science 241:1658-1661 (1988)). Furthermore, it has been found that antisera from patients with Lambert-Eaton Myasthenic Syndrome (an autoimmune disease of neuromuscular transmission) could inhibit calcium channel currents (Kim et al., Science 239:405-408 (1988)). Antisera from some patients with Isaacs' Syndrome (acquired neuromyotonia) have antibodies against potassium channels and could increase neuronal excitability, possibly due to blocking of potassium currents (Shillito et al., Ann. Neuol. 38:714-722 (1995)). One monoclonal antibody that was generated against membrane fragments of the eel electroplax attenuates sodium current (Meiri et al., Proc. Natl. Acad. Sci. USA 88:8385-8399 (1986)). Another monoclonal antibody that recognizes the dihydropyridine-binding complex in rabbit muscle transverse tubules inhibits calcium current in a mouse muscle cell line (Morton et al., J. Biol Chem. 263:613-616 (1988)). However, in all these cases, the binding sites on the channel proteins was not clear.
The challenge now is to pin-point the underlying molecular identities (ion channel proteins) responsible for the observed ionic currents in native cells and to define their physiological functions. Although genetic manipulation with targeted deletion of ion channel genes would be helpful, the interpretation of results could be complicated by functional redundancy and developmental abnormalities. Some ion channel blockers are available, but they usually affect a group of ion channels and, thus, lack specificity towards one specific channel protein. These blockers were found empirically, either by clinical use or by broad functional screening, rather than by rational design. The present invention is directed to overcoming these deficiencies.